Abstract Liquid biopsy tests based on cfDNA analysis have garnered a lot of interest, and show promise for being able to determine the somatic mutational status of patients undergoing cancer treatment. For a significant fraction of advanced stage patients, good agreement has been demonstrated between cfDNA and solid tumor variants. Despite these exciting results, significant room for improvement remains. Even when using the leading cfDNA CLIA test (Guardant Health) and testing advanced stage III and IV patients, about 30% of samples don?t have sufficiently high prevalence variants to be detected. This leading test has demonstrated a detection limit of as low as 0.3% allelic frequency (Guardant Health), but its specificity has only been demonstrated to be high down to 2%. Sensitivity and specificity improvements will enable both test providers and individual genomic test labs to routinely employ liquid biopsy tests across broad patient populations, and earlier in the disease process. The goal of this project is to develop a liquid biopsy workflow using ERASE-Seq, an ultra-sensitive somatic variant caller. This is the first variant caller to make variant calls based on intersample data and a background- aware model, which eliminates recurrent artifacts. A variant event matrix is assembled, containing the number of occurrences in multiple sample and background runs for each variant. A statistical test then determines the confidence score for presence of each variant over background, eliminating stochastic errors that contribute to false positive calls. The initial development and validation was performed using gDNA on a variety of different sequencing technologies. High sensitivity detection of variants in sequencing data down to 0.1% allelic frequency was shown, with no false positives detected across a full targeted somatic panel (>30kb). This performance is superior to any molecular barcoding panels currently available and liquid biopsy CLIA tests for which sensitivity and specificity data is available. If successful, this grant will develop a universal tool for variant detection in cfDNA samples, providing order of magnitude gains in sensitivity for a variety of different workflows and panels, without the added complications associated with molecular barcodes. All users will have to do to use this tool is use the ERASE-Seq targeted amplification kit and deliver fastq data to our ERASE caller software.